Screening for Protein-Protein Interactions in the Yeast Two-Hybrid System in Embryonic Stem Cells

The two-hybrid system (THS) (1 ) is a molecular genetic screen that detects protein-protein interactions. The protein specified by the yeast GAL4 gene activates the transcription of genes involved in galactose metabolism. It has two functional domains, a DNA binding domain, Gal4_BD , and a transcriptional activating domain, Gal4_AD , which interact with DNA sequences in the promoter regions of GAL1, GAL2 , and GAL7 to stimulate transcription. The screen involves two plasmids; one carries the GAL4 _BD sequence fused, in-frame, to a sequence coding for a “bait” protein, and the other carries GAL4 _AD sequences, fused to “prey” sequences from a cDNA library. The two plasmids are introduced, typically by transformation, into a yeast strain carrying a reporter gene coupled to a GAL1, GAL2 , or GAL7 promoter. If the proteins encoded by the bait and prey sequences interact to allow correct positioning of the Gal4_AD and GaL4_BD moieties, the reporter gene is activated. Transformants are plated on medium that allows the detection of reporter gene activation. The plasmid carrying the GAL4 _AD :cDNA plasmid can be recovered, and the positive cDNA isolated and characterized.